Cathepsin X/Z/P (Cat X) is a lysosomal cysteine protease that exhibits monocarboxypeptidase activity. Increased Cat X expression is associated with cancer and inflammation, although its roles in normal physiology are still poorly understood. It is expressed by antigen-presenting cells such as dendritic cells (DCs). DCs undergo functional changes upon agonism of pattern recognition receptors (e.g., Toll-like receptors) by pathogen-associated molecular patterns such as CpG. Mature DCs can secrete cytokines, present antigens, etc. We hypothesise that Cat X contributes to DC function, but this has not been examined in detail until now.
We used activity-based probes, immunoblotting, and immunofluorescence imaging to measure active and total levels of Cat X in naïve DCs (DC1940) or those stimulated with TLR-1/2, 2/6, 3, 4, 7/8, and 9 agonists (Pam3, FSL-1, Poly I:C, LPS, R-848, and CpG, respectively). Lysosomal and secreted Cat X levels were significantly elevated in response to CpG treatment, and to a much lesser extent with the other agonists.
To examine the impact of Cat X on DC function, we generated Cat X-deficient DC1940 cells using CRISPR-Cas9. Expression of surface markers indicating DC maturation was unaffected by Cat X deficiency. By contrast, we observed a reduction in the secretion of several chemokines and cytokines (CCL3, CCL5, CD14, IL-12 and TNF-α) upon CpG stimulation in Cat X-deficient cells. Cat X-deficient cells exhibited altered processing of cathepsin L, while the activities of other lysosomal proteases were unaffected. We also found that Cat X-deficient cells had impaired antigen presentation.
Collectively, these data indicate that Cat X is strongly upregulated by TLR-9 agonism in DCs. While it may not be essential for DC maturation, Cat X may regulate cytokine secretion, processing of related endolysosomal proteases, and antigen presentation. Future studies will rescue Cat X expression in Cat X-deficient cells to validate the phenotypes discovered.